Hi Aunchaleepon: My guess is that your culture is overgrown. You are using DMEM instead of the recommended EMEM to culture. The DMEM is enriched for growth. Also, the seeding density is high to begin the culture, try starting with fewer cells. The TEER will not change significantly if the cells are too crowded and the color change on the media is another overgrowth indicator.

You should not need to wash the cells in PBS, this will only add to potential contamination or dilution of the culture medium. The cells should be observable under light microscopy at least on the apical surface. Best of luck.

Hello Aunchaleepon Nooplod

I agree with Bethany McGonnigal . I would like to suggest some more points.

1. Try a range of lower seeding densities and observe the TEER values. Seeding high density can inhibit proper differentiation. An excessively high density can cause overcrowding, leading to multilayer growth instead of a single, uniform monolayer. Multilayering can disrupt proper tight junction formation and compromise the integrity needed for high TEER.

2. Caco-2 cells can change characteristics, including TEER values, with higher passage numbers. A high passage number can lead to an unstable or "leaky" monolayer, preventing the TEER from increasing. Use Caco-2 cells from a low passage number, typically between passages 20 and 50, for reliable and reproducible TEER results.

3. Regularly check your monolayers under a phase-contrast microscope for even, uniform, cobblestone-like morphology. An incomplete monolayer or patches of cell death will result in low TEER.

4. While changing the medium every two days is standard, the technique is critical. Abrasive pipetting that touches and damages the delicate monolayer can disrupt tight junctions and prevent TEER from rising. Perform media changes very gently. Aspirate the medium from both the apical (insert) and basolateral (well plate) chambers carefully, avoiding contact with the cell layer. Refill the chambers by dispensing fresh media down the side of the well to minimize turbulence.

5. Although you cultured your cells for 21 days, some Caco-2 protocols require 21 to 25 days for the monolayer to fully polarize and for the TEER to plateau. Depending on the cell line and conditions, you may need to extend your culture period slightly to achieve maximum TEER.

6. Validate your equipment and protocol. Have you properly zeroed the voltohmmeter with a blank insert? Measure the TEER of blank inserts filled with media to ensure your voltohmmeter is functioning correctly and calibrated. Was the probe correctly placed for each measurement?

7. The volume of media in the apical and basolateral chambers is important for proper gas and nutrient exchange. Using too little media can cause waste buildup and pH shifts, potentially shocking and harming the cells.

8. It is recommended to use 20% FBS during the initial culture phase (first 2-3 days) to promote attachment, then reduce it to 10% for the remainder of the culture.

9. Finally, check for microbial contamination as it can negatively affect cell health and monolayer integrity, leading to low TEER.

Hope these suggestions are helpful!

Best,

Thank you, Bethany McGonnigal and Malcolm Nobre for this tip. I’d like to ask if it’s absolutely necessary to measure TEER, since I’m currently facing equipment issues. Are there any alternative methods to confirm that my Caco-2 cells are suitable for the next experiment ?